replication, transcription, translation and its regulation
TRANSCRIPT
Replication, Transcription, Translation and its regulation
By,Abhinava J V
University of Agricultural Sciences, Dharwad
DNA REPLICATIONDNA replication is the
process by which DNA makes a copy of itself during cell division.
It is Semiconservative replication.
Semiconservative replication would produce two copies that each contained one of the original strands and one new strand
InitiationThe first step in DNA replication is to ‘unzip’
the double helix structure of the DNA molecule at Origins of Replication region & it is mediated by is mediated by DnaA & helicase in Prokaryotes & DNA polymerase α in Eukaryotes.
The separation of the two single strands of DNA creates a ‘Y’ shape called a replication ‘fork’. The two separated strands will act as templates for making the new strands of DNA.
SSB protein prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing and DNA gyrase is needed to relieve the stress helicase and in eukaryotes it is maintained by Topoisomerase.
Before new DNA strands can form, there must be RNA primers present to start the addition of new nucleotides
Primase is the enzyme that synthesizes the RNA Primer
DNA polymerase can then add the new nucleotides
Elongation DNA polymerase uses each strand as
a template in the 3’ to 5’ direction to build a complementary strand in the 5’ to 3’ direction by adding the complementary dNTP’s.
One of the strands is oriented in the 3’ to 5’ direction, this is the leading strand The other strand is oriented in the 5’ to 3’ direction, this is the lagging strand.
As a result of their different orientations, the two strands are replicated differently.
leading strandDNA polymerase slides along the leading
strand in the 3’ to 5’ direction synthesizing the matching strand in the 5’ to 3’ direction.
The RNA primer is degraded by RNase H and replaced with DNA nucleotides by DNA polymerase, and then DNA ligase connects the fragment at the start of the new strand to the end of the new strand
lagging strandChunks of DNA, called Okazaki fragments, are
then added to the lagging strand also in the 5’ to 3’ direction & it occurs in discontinuous manner
The RNA primers are degraded by RNase H and replaced with DNA nucleotides by DNA polymerase
DNA ligase connects the Okazaki fragments to one another
TerminationTermination of DNA replication in
Prokaryotes is completed through the use of termination sequences and the Tus protein. These sequences allow the two replication forks to pass through in only one direction, but not the other.
In eukaryotic cells the end replication problem of telomere regions is handled by telomerase.
Telomeres extend the 3' end of the parental chromosome beyond the 5' end of the daughter strand.
This 3' addition provides a template for extension of the 5' end of the daughter strand by lagging strand DNA synthesis.
RegulationIn Prokaryotes, regulation of DNA replication
is achieved through several mechanisms. Mechanisms involve the ratio of ATP to
ADP, of DnaA to the number of DnaA boxes and the hemimethylation and sequestering of OriC.
In Eukaryotes, DNA replication is controlled within the context of the cell cycle.
As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints.
TRANSCRIPTIONTranscription is the synthesis of a single-
stranded RNA molecule using the DNA template.
Regulated by gene regulatory elements within each gene.
RNA is transcribed 5’ to 3’ from the template (3’ to 5’).
Similar to DNA synthesis, except: NTPs instead of dNTPs (no deoxy-) No primer No proofreading Adds Uracil (U) instead of thymine (T) RNA polymerase
InitiationRNA polymerase combines with sigma
factor (a polypeptide) to create RNA polymerase holoenzyme which recognizes promoters and initiates transcription.
RNA polymerase holoenzyme binds promoters and untwists DNA
It binds loosely to the -35 promoter and then binds tightly to the -10 promoter and untwists the DNA
ElongationElongation of RNA chain takes place by the
addition of ribonucleotides to the 3'-end of the RNA so that the RNA chain grows in 5'-3' direction.
As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it .
Since the base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components, RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.
TerminationIn prokaryotes, termination of
transcription is brought about by certain termination signals on DNA called terminators (these are DNA sequences).
1. Intrinsic termination- In the stem of the RNA, there is a stretch of G-C reach segment. The G-C reach segment results in a hair-pin loop formation in the RNA stem.
• As a result the weak association between A-U in the long stretch of termination sequence break and the RNA is released.
2. Extrinsic termination- rho
protein dependent.Rho factor binds to the 5'-end of
nascent m-RNA and scans down along the length of m-RNA until it reaches the termination point.
At termination point when the transcription slows down rho breaks ATP and utilizes that energy to denature the RNA-DNA hybrid so that the RNA is released from the bubble.
In Eukaryotes the termination
process differs for each of the three RNA polymerases.
Most of eukaryotes possess robust methods of regulating transcription initiation on a gene-by-gene basis.
The transcription of a gene can be regulated by cis-acting elements within the regulatory regions of the DNA, and trans-acting factors that include transcription factors and the basal transcription complex.
Post-Transcriptional ModificationDNA transcription occurs in a cell's
nucleus. The RNA that is synthesized in this
process is then transferred to the cell's cytoplasm where it is translated into a protein.
In prokaryotes, the RNA it is ready for translation into a protein.
Eukaryotic RNA from DNA transcription, is not immediately ready for translation and it needs Post-Transcriptional Modification.
RNA splicing - A two-step reaction in which introns are removed from a primary RNA transcript and exons are joined together to form mature mRNA.
5' Capping - occurs in cell nucleus, the addition of a GTP molecule to the 5' end of a primary RNA transcript forming a 5'-5' linkage between the two.
Poly A tail - occurs in cell nucleus, A string of up to 500 adenines added to the 3' end of primary RNA transcripts. Addition catalyzed by the enzyme poly (A) polymerase that recognizes the sequence AAUAAA.
RegulationProkaryotic transcription is regulated by
three main sequence elements.Promoters are elements of DNA that
may bind RNA polymerase and other proteins for the successful initiation of transcription directly upstream of the gene.
Operators recognize repressor proteins that bind to a stretch of DNA and inhibit the transcription of the gene.
Positive control elements that bind to DNA and incite higher levels of transcription
In Eukaryotes 3 mechanisms. Were involvedControl over polymerase access to the gene. This includes the functions of histone, remodeling enzymes, transcription factors, enhancers and repressors, and many other complexes.
Productive elongation of the RNA transcript. Once polymerase is bound to a promoter, it requires another set of factors to transcribing the RNA.
Termination of the polymerase A number of factors which have been found to control how and when termination occurs, which will dictate the fate of the RNA transcript.
TRANSLATIONThe mechanism for translating
messenger RNA into protein in eukaryotic cells is basically the same as in prokaryotes. That is, messenger RNA is read by ribosomes.
The ribosome and its subunits are larger in eukaryotes. 40S and 60S subunits combine to form a functional 80S ribosome. In prokaryotes, the analogous particles are 30S & 50S subunits combine to form a functional 70S.
InitiationInitiation of translation involves
the assembly of the ribosomal subunits and it is mediated by the Initiation factors.
Prokaryotes- IF1, IF2, and IF3Eukaryotes – eIF1, eIF2, eIF3, eIF4,
eIF5, eIF6In Eukaryotes Initiation usually
involves the interaction of certain key proteins with a special tag bound to the 5' cap.
The ribosome has three sites: the A site, the P site, and the E site.
The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fMet-tRNAf
Met, which enters at the P site).
The P site is where the peptidyl tRNA is formed in the ribosome.
The E site which is the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.
The smaller subunit binds to the mRNA in upstream purine-rich region of the AUG (initiation codon).
ElongationElongation of the polypeptide
chain involves addition of amino acids to the carboxyl end of the growing chain.
Elongation starts when the fMet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind.
Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain.
The growing polypeptide connected to the tRNA in the P site is detached and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site. This process, known as peptide bond formation, is catalyzed by a ribozyme.
The newly formed peptide in the A site tRNA is known as dipeptide and the whole assembly is called dipeptidyl-tRNA.
The tRNA in the P site minus the amino acid is known to be deacylated.
In the final stage of elongation, called translocation, the deacylated tRNA (in the P site) and the dipeptidyl-tRNA (in the A site) along with its corresponding codons move to the E and P sites, respectively, and a new codon moves into the A site. This process is catalyzed by elongation factor G (EF-G).
The deacylated tRNA at the E site is released from the ribosome during the next A-site occupation by an aminoacyl-tRNA it is facilitated by EF-Tu.
TerminationTermination occurs when termination
codons (UAA, UGA, or UAG )moves into the A site.
These codons are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons).
These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome.
RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process
RegulatationTranslation is regulated on many
levels. The joining of the two ribosomal subunits can be blocked by RsfS.
RsfS binds to L14, a protein of the large ribosomal subunit, and thereby blocks joining of the small subunit to form a functional 70S ribosome, slowing down or blocking translation entirely.
RsfS proteins are found in almost all eubacteria (but not archaea) and homologs are present in mitochondria and chloroplasts (where they are called C7orf30 and iojap, respectively).
RNA interference (RNAi) : On the other hand to control the synthesis of Proteins from the mRNA, DNA synthesis the complementary RNA to that mRNA & it is called as RNA interference.
It is done by small RNA called as microRNA (miRNA) and small interfering RNA (siRNA)
It forms the RNA-RNA Hybrid and thus involves in the cleavage of mRNA was occur by the enzyme called Dicer.
